Potential novel mechanism for Axenfeld-Rieger syndrome: deletion of a distant region containing regulatory elements of PITX2

Abstract

Purpose: Mutations in PITX2 are associated with Axenfeld-Rieger syndrome (ARS), which involves ocular, dental, and umbilical abnormalities. Identification of cis-regulatory elements of PITX2 is important to better understand the mechanisms of disease.

Methods: Conserved noncoding elements surrounding PITX2/pitx2 were identified and examined through transgenic analysis in zebrafish; expression pattern was studied by in situ hybridization. Patient samples were screened for deletion/duplication of the PITX2 upstream region using arrays and probes.

Results: Zebrafish pitx2 demonstrates conserved expression during ocular and craniofacial development. Thirteen conserved noncoding sequences positioned within a gene desert as far as 1.1 Mb upstream of the human PITX2 gene were identified; 11 have enhancer activities consistent with pitx2 expression. Ten elements mediated expression in the developing brain, four regions were active during eye formation, and two sequences were associated with craniofacial expression. One region, CE4, located approximately 111 kb upstream of PITX2, directed a complex pattern including expression in the developing eye and craniofacial region, the classic sites affected in ARS. Screening of ARS patients identified an approximately 7600-kb deletion that began 106 to 108 kb upstream of the PITX2 gene, leaving PITX2 intact while removing regulatory elements CE4 to CE13.

Conclusions: These data suggest the presence of a complex distant regulatory matrix within the gene desert located upstream of PITX2 with an essential role in its activity and provides a possible mechanism for the previous reports of ARS in patients with balanced translocations involving the 4q25 region upstream of PITX2 and the current patient with an upstream deletion.

Figure 1.

Schematic drawing of the genomic organization/isoforms of the (A) human PITX2 and (B) zebrafish pitx2 genes. PITX2/pitx2 exons are indicated as numbered boxes. Conserved elements are shown as black boxes/lines and are marked CE1, CE2, CE3, and CE4-CE13. Exonic sizes are indicated at the top intronic sizes are shown at the bottom of the numbered boxes and Distance to the distant element CE4 from the first exon and size of the genomic region containing CE4 to CE13 elements are indicated next to the brackets encompassing the corresponding regions (see Supplementary Table S1, http://www.iovs.org/lookup/suppl/doi:10.1167/iovs.10-6060/-/DCSupplemental, for more details). Position of the pitx2 probe used in in situ hybridization experiments is indicated.

Figure 2.

Expression of the zebrafish pitx2 gene during development. Images of whole mount embryos (A–L) and cryosections (M–O) are presented. Lateral (A, D, G, J), ventral (B, E, H, K), and dorsal (C, F, I, L) views are shown for every stage. Developmental stages are indicated on the left (whole mount) or in the lower right (cryosections). Note the expression in the periocular mesenchymal (pm) cells of neural crest origin that migrated into the anterior segment of the eye in 24- to 48-hpf embryos (A–F, M) and continuing strong expression in the developing anterior segment structures (ac), specifically the ventral canal, in 72- to 120-hpf embryos (G–L, N, O). Expression in the developing brain (b, brain; di, diencephalon; mb, midbrain), around the oral cavity (oc), and in the pharyngeal arches (pa) was also observed at the examined stages.

Figure 3.

Reporter expression mediated by conserved elements identified in the PITX2 region. GFP expression associated with pitx2 promoter region only (A), and pitx2 promoter region with additional elements CE2 (B–D), CE3 (E, F), CE6 (G), CE9 (H, I), CE12 (J), CE13 (K, L), CE5 (M), CE7 (N), CE10 (O), and a combination of CE5, CE7, and CE10 (P) in 36- to 80-hpf embryos. Note the strong expression in the developing brain in 36-hpf (B) and 48-hpf embryos for CE2 (C, D) and 48-hpf embryos for CE3 (E, F), CE6 (G), CE9 (H), and CE12 (J). For CE9, expression in the developing brain weakened by 80 hpf, whereas robust expression in the heart region became evident (I). CE13 was associated primarily with expression during heart and craniofacial development in 24-hpf (K) and 48-hpf (L) embryos. CE5, CE7, and CE10 regions appeared to be associated with ocular expression that could be easily observed in 48-hpf embryos (M–P). b, brain; di, diencephalon; h, heart; mb, midbrain; oc, oral cavity; pm, periocular mesenchyme; sm, skeletal muscles (trunk).

Figure 4.

Reporter expression in Tg(-2.6pitx2-CE4:GFP) permanent transgenic line. (A–E) GFP fluorescence images. (F–H) Images of in situ hybridization performed using Tg(-2.6pitx2-CE4:GFP) embryos and GFP antisense riboprobe. Developmental stages are indicated in the lower right of every image. Note the strong expression in the periocular mesenchymal (pm) cells of neural crest origin that migrated to the anterior segment region (A, B) and expression in the developing brain (A–G) and around the oral cavity (C–G), pharyngeal arches (E), and the anterior segment of the eye (G, H). ac, anterior segment of the eye; b, brain; di, diencephalon; e, eye; mb, midbrain; oc, oral cavity; pa, pharyngeal arches; pm, periocular mesenchyme; re, retina.

Figure 5.

Images of patient 1 with Axenfeld-Rieger syndrome. (A) Facial photograph showing maxillary hypoplasia, thin upper lip, and broad nasal bridge. (B) Left eye with corectopia. (C) Right eye with posterior embryotoxon. (D) Dental anomalies, including maxillary hypodontia. (E) Redundant periumbilical skin.

Figure 6.

Identification of 4q25–26 deletion in a patient with Axenfeld-Rieger syndrome. (A) Genome Browser view of the region of deletion in patient 1. The deleted region is indicated by a black rectangle, and the previously reported ARS translocations are indicated by with wavy arrows. The shaded area is shown in more detail in B. (B) Enlargement of the region containing PITX2 and the upstream gene desert with regulatory elements (shaded area). Affymetrix Genotyping Console (version 3.0.2.) representation of the array data for patient 1 is shown. Top: 0 to 4 indicate copy number, with 2 corresponding to diploid and 1 (arrow) to haploid states. Positions of TaqMan CNV probes used to confirm Affymetrix array data are shown as black circles and numbered 1 to 6. Position of the patient 1 deletion and translocation breakpoints previously reported in ARS patients are indicated with a dark gray rectangular box and wavy arrows, correspondingly. Regions of genomic variation (Database of Genomic Variants) in this region, as appear in the Genotyping Console, are shown as gray boxes, whereas conserved elements (CE1-CE13) are indicated as black boxes and are numbered 1 to 13; positions of PITX2 and C4ORF32 and chromosomal band q25 are shown as they appear in Genotyping Console. The image clearly demonstrates that no deletions similar in size to the one identified in patient 1 or involving conserved elements have been reported as normal variation. The region between these two transcribed units, PITX2 and C4ORF32, is devoid of any genes and therefore represents a gene desert. (C) Copy number variation analysis with TaqMan probes within PITX2 (P1–P3) and upstream region (P4–P6) in a family with Axenfeld-Rieger syndrome. Pedigree is shown on the left. Note proband with ARS and unaffected parents. Summary of qPCR analysis (right) shows normal diploid state for all six probes in unaffected parents and diploid state for probes P1 to P3 but haploid state for probes P4 to P6 for the proband (arrow).